ATP-synthase terms leaking onto flagellar and T3SS export ATPases

Part of TreeGrafter Inference Evaluation. Opened 2026-09-27.

Bottom line

• Almost every FliI / SctN protein in UniProt is annotated as an ATP synthase.
  These are the ATPases that power flagellar and injectisome (type III) protein
  export. They are paralogs of the F1-ATP synthase β subunit, and they hydrolyse ATP
  rather than make it. Of the 11,205 UniProtKB entries in their family (InterPro
  IPR005714), 97% carry GO:0046933 proton-transporting ATP synthase activity,
  rotational mechanism and 97% carry GO:0015986 proton motive force-driven ATP
  synthesis. Among the 29 reviewed Swiss-Prot entries, all 29 carry GO:0015986
  and 20 carry GO:0046933 (counts).
• One misplaced PAINT node explains the synthase terms. Both IBDs (for
  GO:0046933 and GO:0045259 proton-transporting ATP synthase complex) sit on
  PANTHER:PTN008558586 in PTHR15184. In the live PANTHER tree that node is a
  duplication. Its two children are the F1-β clade PTN008558588, which holds
  every seed, and the FliI/SctN export-ATPase clade PTN000390097 (SF9 SPI-1, SF62
  SPI-2, SF81 flagellar) (placement).
• PAINT IBA passes GO:0046933 to 28 proteins with the FliI/SctN signature
  IPR005714 in the reference genomes, plus a Chlamydia FliI (O84722) that lacks
  the signature (targets). They
  include Salmonella FliI, InvC and SsaN and E. coli FliI.
• TreeGrafter then passes GO:0046933 to every grafted FliI/SctN. All five graft
  nodes seen in our reviews descend from PTN000390097.
• The IBD was placed one node too deep. TreeGrafter faithfully reproduces the
  error rather than causing it.
• Two more pipelines add errors of their own:
• InterPro2GO maps the SctN-specific entry IPR013380 to GO:0046961 proton-
  transporting ATPase activity, rotational mechanism and to GO:0006754 ATP
  biosynthetic process.
• It maps the shared F1/V1/A1 N-terminal domain IPR004100 to GO:1902600 proton
  transmembrane transport and GO:0046034 ATP metabolic process.
• GO's logical inference (GO_REF:0000108) then turns each wrong molecular
  function into a wrong process: GO:0046933 → GO:0015986 and GO:0046961 →
  GO:1902600.
• Nine full reviews reject every such row: 35 rows, 31 REMOVE and 4
  MARK_AS_OVER_ANNOTATED, with none accepted
  (per-row table).

How this was found

It came out of a first-principles completeness test. A process every organism
needs gives an independent truth set: "does this proteome encode a complete F-type
ATP synthase?". The test asks how well plain GO annotation recovers it
(completeness_*.py, results).

• Proteomes: 15,525 bacterial reference proteomes with BUSCO completeness ≥ 95%.
  Of these, 14,959 encode all eight F-type subunit families.
• Accuracy: "has a GO:0046933 protein" scores precision 0.967 and recall 0.976
  against that truth.
• False negatives: new TrEMBL entries that have InterPro cross-references but no
  GO annotations yet.
• False positives: mostly V/A-type ATPases and FliI/SctN. The FliI/SctN false
  positives are genuine annotation errors, and they are the subject of this page.
• Genuine absences: the complex is missing from phytoplasmas, and V/A-type
  ATPases replace it in many Clostridia, Spirochaetia and Bacteroidota.

Why the terms are wrong

The reviews give the evidence with verbatim quotes. In brief:

• FliI/SctN are hydrolases. They are soluble, peripheral ATPases that dock
  export chaperone–substrate complexes, release the chaperones, and unfold and hand
  on the substrates. Their EC is 7.4.2.8, protein-secreting ATPase.
• FliI's ATPase is not an F-type activity. It is insensitive to inhibitors of
  F-, V- and P-type ATPases (PMID:8943245).
• Protons move through the export gate, not the ATPase. The export apparatus
  does use the proton motive force, but the flux runs through the membrane export
  gate, a proton–protein antiporter whose Salmonella FlhA subunit acts as the ion channel (PMID:21934659,
  PMID:29946050). CCCP sensitivity of Ysc secretion reflects this (PMID:15213145),
  not proton transport by SctN.
• The resemblance to F1 is real but does not make them synthases. FliJ/SctO is
  γ-stalk-like, and the FliI₆–FliJ ring resembles F1 (PMID:21278755, PMID:17202259).
  But the ring still hydrolyses ATP and has no Fo sector to couple to.

Reviews

Actions by pipeline:

• The four over-annotations are all GO:0046034 ATP metabolic process. It is
  literally true of an ATPase but adds nothing beyond GO:0016887.
• What replaces the wrong terms. The correct molecular function, GO:0008564
  protein-exporting ATPase activity, is kept where it was already in GOA and
  added (NEW) where it was missing.

Other errors the reviews found along the way:

• UniProt naming. The flagellar FliI entries (P26465, P52612, P0CAT8, Q9ZJJ3)
  are named "Flagellum-specific ATP synthase" with EC 7.1.2.2, the
  H⁺-transporting synthase EC, and keep a 1991 "proton translocase" hypothesis in
  their FUNCTION text (PMID:1646201). The injectisome SctN entries already use
  EC 7.4.2.8.
• SsaN host-cell locations. Two AgBase IMP rows place SsaN in host cell
  cytoplasm and host cell membrane. The paper fractionates bacterial cells, and
  the effector, not the ATPase, is what reaches the host (see
  SALTY/sctN2).

Where this sits among the TreeGrafter failure modes

• Unlike most of the failure modes, this is not a problem with the term attached
  to the graft node, nor a mis-graft. The TreeGrafter placements are correct: every
  graft node is inside the FliI/SctN clade.
• The error comes in from PAINT. The ancestral IBD sits on the duplication node,
  so it covers both paralog clades. This is a fifth pattern: inherited PAINT
  over-placement. Fixing the IBD fixes the TreeGrafter output with no change to
  TreeGrafter.
• Earlier reviews did not catch it. The PTHR15184 family review
  (interpro/panther/PTHR15184/PTHR15184-review.yaml) had rated the PTN008558586
  assertions SOUND, having checked the node against its F1-β descendants only. It
  now records both as TOO_DEEP, lists SF9, SF62 and SF81 as diverged
  subfamilies, and scopes GO:0046933 to the F1-β subfamilies and GO:0008564 to
  the export-ATPase subfamilies (updated 2026-09-27).
• Not in the frozen headline figures. These rows postdate the frozen 2026-09-06
  snapshot, so they are absent from the tables on the main TreeGrafter page.

Suggested upstream fixes

1. PANTHER/PAINT: move the GO:0046933 and GO:0045259 IBDs from
   PTN008558586 to PTN008558588, or add an IRD/NOT at PTN000390097. This one
   change fixes both the IBA rows and every TreeGrafter row.
2. InterPro2GO:
3. Drop GO:0046961 and GO:0006754 from IPR013380. That entry is SctN-specific,
   so the mapping is wrong for every member.
4. Suppress IPR004100 → GO:1902600 / GO:0046034 when IPR005714 also matches.
5. Drop the stale IPR005714 → GO:0009058 biosynthetic process mapping.
6. All five are recorded in the InterPro mapping review
   (mapping set).
7. UniProt: rename the flagellar FliI entries to a flagellar export ATPase, use
   EC 7.4.2.8, and drop the "proton translocase" clause.

Reproduce

uv run --with requests projects/TREEGRAFTER/rotary_atpase/leak_scan.py          # UniProt counts + PTN008558586 IBA targets
uv run --with requests projects/TREEGRAFTER/rotary_atpase/node_placement.py     # PANTHER tree placement of IBD and graft nodes
uv run --with pyyaml   projects/TREEGRAFTER/rotary_atpase/review_actions.py     # per-row reviewer actions from the gene reviews
uv run --with requests projects/TREEGRAFTER/rotary_atpase/completeness_fetch.py # bacterial F-type completeness (large; to ./cache)
python3 projects/TREEGRAFTER/rotary_atpase/completeness_analyze.py > projects/TREEGRAFTER/rotary_atpase/completeness_results.txt

The UniProt, QuickGO and PANTHER queries run against live services, so the counts
will drift between releases. The committed TSVs were generated on 2026-09-27.